DELETIONS IN THE 3RD INTRACELLULAR LOOP OF THE THYROTROPIN RECEPTOR -A NEW MECHANISM FOR CONSTITUTIVE ACTIVATION

Gain-of-function mutations of the thyrotropin receptor (TSHR) gene hav e been invoked as one of the major causes of toxic thyroid adenomas. I n a toxic thyroid nodule, we recently identified a 9-amino acid deleti on (amino acid positions 613-621) within the third intracellular (i3) loop of the TSHR resulting in constitutive receptor activity. This fin ding exemplifies a new mechanism of TSHR activation and raises new que stions concerning the function of the i3 loop. Because the i3 loop is thought to be critical for receptor/G protein coupling. Thus, various deletion mutants were generated and functionally characterized. We ide ntified an optimal deletion length responsible for constitutive activi ty. If the number of deleted amino acids was reduced, elevated basal c AMP accumulation was found to be concomitantly diminished. Expansion o f the deletion dramatically impaired cell surface expression of the re ceptor. Shifting the deletion toward the N terminus of the i3 loop res ulted in unaltered strong constitutive receptor activity. In contrast, translocation of the deletion toward the C terminus led to significan tly reduced basal cAMP formation, most probably due to destruction of a conserved cluster of amino acids. In this study, we show for the fir st time that amino acid deletions within the i3 loop of a G protein-co upled receptor result in constitutive receptor activity. In the TSHR, 75% of the i3 loop generally assumed to play an essential role in G pr otein coupling can be deleted without rendering the mutant receptor un responsive to thyrotropin. These findings support a novel model explai ning the molecular events accompanying receptor activation by agonist.